Recent human genetic analysis has found that mutations in the cation/proton exchangers NHE6 and NHE9 both result in autism. Little is known about these two closely related proteins or their function in neurons. Interestingly, recent work from the Edwards lab has identified a novel NHE activity on synaptic vesicles. This activity promotes the uptake of excitatory neurotransmitter glutamate into vesicles by converting the vesicle pH gradient (?pH) into membrane potential (??). Furthermore, preliminary work has localized NHE6 and NHE9 to presynaptic boutons and in particular synaptic vesicles, suggesting that these isoforms are responsible for the observed activity. The uptake of different transmitters depends to varying extents on either ?pH or ??, and hence loss of NHE6 or NHE9 would be predicted to have different effects on different transmitters. The hypothesis of this proposal is that NHE6 and 9 convert ?pH into ? ? and thus influence the filling of synaptic vesicles with neurotransmitter, with global synaptic effects that might underlie the severe phenotype observed in patients with mutations in NHE6 or 9 and perhaps other forms of autism. This proposal seeks to characterize the role of NHE6 and NHE9 at multiple physiological levels: synaptic vesicle function, neural transmission and behavior. At the level of synaptic vesicle function, I will first determine whether NHE6 and 9 target to distinct subsets of synaptic vesicles through immunoisolation of synaptic vesicles containing NHE6 and 9 from mouse brain, and characterization of the respective synaptic vesicle proteins. Next, synaptic vesicles isolated from the brains of NHE6 knock-out (KO) and NHE9 conditional KO (cKO) mice will be used to assess the functional role of these two isoforms by measuring the effect of cations (K+ and Na+) on vesicle pH using acridine orange, on membrane potential using oxonol V and on transmitter uptake by radiotracer flux assay. The pH of synaptic vesicles will be measured in live neurons through the culture of primary dissociated KO neurons. At the level of synaptic transmission, analysis of the spontaneous and evoked release of glutamate, GABA and dopamine in slices from KO mice will indicate the role of NHE6 and 9 in quantal size, transmitter release and/or the postsynaptic response. Finally, this proposal seeks to characterize the specific autistic behaviors that result from loss of NHE9, focusing on the three main diagnostic criteria for autism: sociability, repetitive behaviors and communication skills. This work will thus test the hypothesis that the roles of NHE6 and 9 in synaptic vesicle filling, neurotransmitter release and synaptic transmission give rise to the effects on behavior. Ultimately, the correlation of physiological defects with autistic behavior has great potential to elucidate the systems as well as molecular basis for autism, which would in turn implicate these mechanisms as targets for rational, therapeutic intervention.